Stabilization of Labile Analytes in Reference Materials

ABSTRACT

Provided herein are assay control materials comprising stable analytes and lyophilized unstable analytes, and methods of making and using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Appl. No.61/819,758 filed May 6, 2013, the disclosure of which is incorporatedherein in its entirety.

BACKGROUND OF THE INVENTION

Commercially available quality control materials are routinely used inthe clinical diagnostics laboratories to monitor the precision andaccuracy of both manual and automated clinical test methods andprocedures. Development of multi-analyte and multi-level quality controlmaterials, however, is very complex due to differences in chemical andphysical properties of the biomarkers/analytes in the control (molecularweight, solubility, reactivity, stability, interferences from otheranalytes, etc).

Multi-analyte and multi-level quality control materials typically havestability and performance limitations for at least one analyte becauseconditions which may improve stability of one analyte may be detrimentalto stability of another. For example, Randox Liquid Chemistry PremiumPlus has a performance limitation for stability of total and directbilirubin, and the instruction pamphlet states that total and directbilirubin values gradually decrease during the product shelf life. Inaddition, the instructions for MAS® ChemTRAK® Liquid Unassayed ChemistryControl (Thermo Scientific) states that bilirubin may decrease over theproduct shelf life and phosphorus, salicylate, and triglycerides mayincrease over product shelf life.

Available urine chemistry quality controls can suffer from the poorstability of creatinine. This biomarker is a clinically importantanalyte in commercially available urine chemistry controls because it isused to evaluate the renal function. An example of a urine chemistryquality control with poor stability for creatinine is MAS® UrichemTRAK®(Thermo Scientific). The instruction for use for this quality controlstates that creatinine values may decrease over the product shelf life.

BRIEF SUMMARY OF THE INVENTION

Provided herein are stable multianalyte quality control materials formonitoring the performance of various diagnostic testing methodologiesin clinical laboratories. The presently described multianalyte controlmaterials address the problem of unstable control solutions, can quicklyand easily be prepared at the point of use, and can meet the qualitycontrol needs of a lab with regards to the stability of analytes. Inaddition to meeting the stability requirements of a quality controlmaterial, the presently described materials meet other requirements,such as responding in the same manner to the same analytical variancesfound in patient samples, by using human base matrices.

Provided are multianalyte control kits comprising: a first container(e.g., tube, ampoule, or vial) holding at least one unstable controlanalyte, wherein the at least one unstable control analyte isstabilized, e.g., by drying or lyophilization. In some embodiments, thefirst container holds 2, 3, 4, or 5 unstable analytes in stabilizedform. In some embodiments, the first container includes more than oneanalyte, e.g., stable or unstable, and the analytes are all instabilized form, e.g., dried or lyophilized. In some embodiments, thefirst container holds at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 1-5,3-10, 10-15, 10-20, or more analytes in stabilized form. In someembodiments, the at least one unstable analyte is lyophilized in theform of beads (e.g., microbeads or microspheres). In some embodiments,the bead has a diameter of 1-15 mm. In some embodiments, the at leastone unstable analyte is selected from the group consisting ofcreatinine, bilirubin, salicylate, triglyceride, alanineaminotransferase (ALT), alkaline phosphatase, high density lipoprotein,pseudocholinesterase, folate, and homocysteine.

In some embodiments, the kit further comprises a second containerholding at least one stable control analyte. In some embodiments, the atleast one stable analyte is in solution. In some embodiments, thesolution is a base matrix solution derived from a biological sample,e.g., processed urine, plasma, serum, saliva, synovial fluid, lymph,milk, mucus, CSF, cell lysate, or tissue culture supernatant. In someembodiments, the at least one stable control analyte is selected fromthe group consisting of amylase, calcium, chloride, glucose, hCG,magnesium, microalbumin, phosphorus, protein, sodium, urea nitrogen, anduric acid. In some embodiments, the at least one stable analyte is insolution at osmolality of 50-1000 mmol/kg (e.g., 100, 150, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950,200-800, 250-750, 400-600, etc.). In some embodiments, the at least onestable analyte is in solution at pH 4-9 (e.g., about 4-5, about 5-6,about 6-7, about 7-8, or about 8-9). In some embodiments, the basematrix solution comprises at least one component selected from: PEG,HSA, BSA, human hemoglobin, protease inhibitor, chelating agent, buffer,salt, antioxidant (or antioxidant enzyme), cryoprotectant, surfactant,and antibiotic agent (e.g., sodium azide, ciprofloxacine,chloramphenicol, gentamicin, amikacin, tobramycin, and amphotericin B).

In some embodiments, the kit further comprises a third container holdinga solution for resuspending the at least one unstable analyte in thefirst container. In some embodiments, the third container holds a basematrix solution derived from a biological sample. In some embodiments,the solution has an osmolality of 50-1000 mmol/kg. In some embodimentsthe solution has a pH of 4-9. In some embodiments, the base matrixsolution comprises at least one component selected from: PEG, HSA, BSA,human hemoglobin, protease inhibitor, chelating agent, buffer, salt,antioxidant (or antioxidant enzyme), cryoprotectant, surfactant, andantibiotic agent (e.g., sodium azide, ciprofloxacine, chloramphenicol,gentamicin, amikacin, tobramycin, and amphotericin B). In someembodiments, the multianalyte control kit includes at least 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 1-5, 3-10, 10-15, 10-20, or more analytes.

Further provided are multilevel, multianalyte control kits comprising afirst set of containers, wherein each of the first set of containersholds a different amount of at least one unstable analyte, and whereinthe at least one unstable control analyte is stabilized, e.g., by dryingor lyophilization. In some embodiments, the kit further comprises asecond set of containers, wherein each of the second set of containersholds a different amount of at least one stable analyte. One of skillwill understand that multilevel, multianalyte controls are typicallydesigned so that all of the analytes at a given level are at acomparatively high amount, while all the analytes at another level areat a comparatively low amount, etc., depending on how many levels arecontemplated. In some embodiments, the multilevel, multianalyte kit is abilevel or trilevel kit. In some embodiments, the multilevel,multianalyte control kit includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 1-5, 3-10, 10-15, 10-20, or more analytes.

In some embodiments, the multianalyte control kit used for an assaytesting urine chemistry, blood serum chemistry, diabetes markers, cancermarkers, immune function, or cardiovascular health.

Further provided are methods for assembling a multianalyte control kitsuch as those described above, comprising: (i) selecting at least oneunstable analyte in the multianalyte control kit and stabilizing (e.g.,drying or lyophilizing) the at least one unstable analyte in a firstcontainer and (ii) adding at least one stable analyte in themultianalyte kit to a second container. In some embodiments, the atleast one unstable control analyte is lyophilized in the form of beads.In some embodiments, the at least one stable analyte is in solution. Insome embodiments, the at least one unstable control analyte is selectedfrom the group consisting of creatinine, bilirubin, salicylate,triglyceride, alanine aminotransferase (ALT), alkaline phosphatase, highdensity lipoprotein, pseudocholinesterase, folate, and homocysteine.

Further provided are methods for preparing a multianalyte control kitsuch as those described above for use in an assay, comprising: (i)resuspending at least one unstable analyte in stabilized (dried orlyophilized) form and (ii) combining the at least one unstable analytewith at least one stable analyte in solution. In some embodiments, theresuspending and combining steps are simultaneous, e.g., the at leastone unstable analyte is resuspended in the solution comprising the atleast one stable analyte. In some embodiments, the resuspending andcombining steps are separate, e.g., the at least one unstable analyte isresuspended in a solution, and then combined with the at least onestable analyte. In some embodiments, the resuspending solution is a basematrix solution. In some embodiments, the at least one unstable analyteand at least one stable analyte are added to a base matrix solution. Insome embodiments, the base matrix solution is a biological sample orderived from a biological sample, e.g., processed urine, plasma, serum,saliva, lymph, milk, mucus, CSF, cell lysate, or tissue culturesupernatant. In some embodiments, the solution comprises at least onecomponent selected from: PEG, HSA, BSA, human hemoglobin, proteaseinhibitor, chelating agent, buffer, salt, antioxidant (or antioxidantenzyme), cryoprotectant, surfactant, and antibiotic agent (e.g., sodiumazide, ciprofloxacine, chloramphenicol, gentamicin, amikacin,tobramycin, and amphotericin B).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Bead to bead variability of creatinine beads (20 beads tested inquadruplicate).

DETAILED DESCRIPTION OF THE INVENTION A. INTRODUCTION

Provided herein are compositions and approaches to address variablestability of analytes within mulianalyte control products. Analytes thatare known to remain stable in solution are provided in a standard,ready-to-use solution, while those that are less stable in the standardsolution are provided in stablized, e.g., lyophilized, form. Thisapproach allows for more reliable results, and extended shelf life ofmultianalyte controls, e.g., for clinical testing facilities.

B. DEFINITIONS

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by a person of ordinaryskill in the art. See, e.g., Lackie, DICTIONARY OF CELL AND MOLECULARBIOLOGY, Elsevier (4^(th) ed. 2007); Sambrook et al., MOLECULAR CLONING,A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor,N.Y. 1989). The term “a” or “an” is intended to mean “one or more.” Theterm “comprise” and variations thereof such as “comprises” and“comprising,” when preceding the recitation of a step or an element, areintended to mean that the addition of further steps or elements isoptional and not excluded. Any methods, devices and materials similar orequivalent to those described herein can be used in the practice of thisinvention. The following definitions are provided to facilitateunderstanding of certain terms used frequently herein and are not meantto limit the scope of the present disclosure.

The term “analyte” refers to a substance to be detected or quantitated.A “mulitanalyte” assay refers to an assay that detects or quantitatesmore than one analyte. Likewise, a multianalyte control will includemore than one analyte in a known (control) amount. A “multilevel”control refers to a plurality of controls, each control containing thesame analyte(s), where the amount of analyte(s) in each control isdifferent. For example, a bilevel control would have two separatecontrols, one with a low level of the included analytes and the otherwith a high level of the included analytes. A trilevel control wouldhave three separate controls, e.g., low level, mid-level, and highlevel.

The term “unstable analyte” can be defined quantitatively or relative toother analytes intended to be used in combination, e.g., in amultianalyte kit. The unstable analyte is one that is degraded (lessdetectable) after a specified time interval at the same storagecondition as the stable analyte(s) by at least 0.5%, 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10% or more. In some cases, an unstable analyte is onethat is degraded (less detectable) after 6 months in solution at roomtemperature by at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% ormore. In some cases, an unstable analyte is one that is degraded (lessdetectable) after 6 months in solution at 4° C. by at least 0.5%, 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more. In some cases, an unstableanalyte is one that degrades faster, e.g., at rate at least 1.2-fold,1.5-fold, 2-fold, 5-fold, or 10-fold faster, or more, than otheranalytes intended to be used in combination when stored in the sameconditions. One of skill will understand that certain classes ofmolecules are likely to fall into this category, depending on storageconditions. Potentially unstable analytes include proteins (e.g.,enzymes, hormones, peptides), organic molecules, and even elements thatbecome less detectable over time when stored in a liquid.

Similarly, the term “stable analyte” can be defined quantitatively orrelatively. Stability is defined in “Evaluation of Stability of In VitroDiagnostic Method Products; Approved Guideline” (Clinical and LaboratoryStandards document EP25A) as the ability of an IVD product to maintainits performance characteristics consistently over time. In some cases, astable analyte does not detectably degrade after a significantproportion of the intended shelf life at the recommended storagecondition, e.g., after 6 or 12 months in solution at room temperature.In some cases, a stable analyte does not detectably degrade after 6 or12 months in solution at 4° C. In some embodiments, a stable analytedegrades at least 1.2-fold, 1.5-fold, 2-fold, 5-fold, or 10-fold lessthan at least one other analyte intended to be used in combination.

As used herein, a “base matrix solution” refers to the solution used tocarry the analyte(s) for detection. A base matrix solution is chemicallysimilar in composition to clinical samples to be tested (e.g., urine,blood, lymph, etc.), includes at least some of the same components, andis in a similar pH range. Typically, components of the clinical samplethat would interfere with detection of analytes, e.g., high endogenouslevels of the analyte, other analytes to be tested, fibrin, or otherprotein or lipid components, are removed from a base matrix solution.

The terms “label,” “detectable moiety,” and like terms refer to acomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, chemical, or other physical means. For example, usefullabels include fluorescent dyes, luminescent agents, radioisotopes(e.g., ³²P, ³H), electron-dense reagents, enzymes (e.g., as commonlyused in an ELISA), biotin, digoxigenin, or haptens and proteins or otherentities which can be made detectable, e.g., by incorporating aradiolabel into a peptide or antibody specifically reactive with atarget analyte. Any method known in the art for conjugating an antibodyto the label may be employed, e.g., using methods described inHermanson, Bioconjugate Techniques 1996, Academic Press, Inc., SanDiego. The term “tag” can be used synonymously with the term “label,”but generally refers to an affinity-based moiety, e.g., a “His tag” forpurification, or a “strepavidin tag” that interacts with biotin.

A “labeled” molecule (e.g., nucleic acid, protein, or antibody) is onethat is bound, either covalently, through a linker or a chemical bond,or noncovalently, through ionic, van der Waals, electrostatic, orhydrogen bonds to a label such that the presence of the molecule may bedetected by detecting the presence of the label bound to the molecule.

A “control” sample or value refers to a sample that serves as areference, usually a known reference, for comparison to a test sample.For example, a test sample can be taken from a test condition, e.g., inthe presence of a test compound or in stabilized conditions, andcompared to samples from known conditions, e.g., in the absence of thetest compound or stabilized condition (negative control), or in thepresence of a known compound or condition (positive control). A controlcan also represent an average value gathered from a number of tests orresults. One of skill in the art will recognize that controls can bedesigned for assessment of any number of parameters. For example, acontrol can be devised to compare signal strength in given conditions,e.g., in the presence of a stabilized (e.g., lyophilized) analyte, inthe absence of a stabilized analyte (negative control), or in thepresence of a known stable analyte (positive controls). One of skill inthe art will understand which controls are valuable in a given situationand be able to analyze data based on comparisons to control values.Controls are also valuable for determining the significance of data. Forexample, if values for a given parameter are variable in controls,variation in test samples will not be considered as significant.

C. FORMATION OF STABILIZED COMPOSITIONS

Unstable analytes can be stored in a stabilized composition such as adry powder. The drying of a solution comprising at least one unstableanalyte can be accomplished using any appropriate method, such as thosedescribed herein, lyophilization, freeze drying, or fluidized beddrying. Lyophilization typically involves inserting the solution into avacuum chamber, or otherwise applying a vacuum to the solution (see,e.g., U.S. Pat. Nos. 7,588,942, 7,354,597, and 7,073,349). In someembodiments, heat or a drying gas is applied. Freeze drying typicallyinvolves spraying a solution through an atomizing nozzle into a cold gasphase such as liquid nitrogen (see, e.g., U.S. Pat. No. 7,007,406,Leuenberger (2002) J. Nano. Res. 4:111). Fluidized bed drying isdescribed, e.g., in U.S. Pat. No. 4,624,058, Sellers et al. J. Pharm.Sci. 90:785, and Frake et al. (1997) Int J. Pharm. 151:75. Once astabilized dry composition is obtained, it can be stored in a sealedcontainer to maintain stability and avoid moisture or contamination.

In some embodiments, the unstable analyte(s) are added in solution tomicrobeads or microspheres that are designed to hold a predeterminedvolume of liquid. The solution of microspheres is then subjected todrying. More than one analyte can be in the solution, so that eachmicrosphere holds more than one analyte. In some embodiments, eachanalyte is added at a known concentration, so that each microsphereholds a known amount of analyte. In some embodiments, stabilizedcompositions are formed with multiple analyte concentrations to obtain amulti-level kit, e.g., with multiple tubes, each representing adifferent amount of analyte(s).

D. SOLUTIONS AND COMPONENTS

Provided herein are solutions for analytes, or for resuspendingstabilized (e.g., dryed or lyophilized) analytes. Such solutions aretypically pH controlled, e.g., with a buffer such as PBS, Tris, orHEPES, so that the pH does not have an overly negative effect on theanalytes in solution. Such solutions also can have stabilizing agentssuch as PEG or HSA. Solutions can have additives such as proteaseinhibitors, chelating agents, buffers, salts, antioxidants (orantioxidant enzymes), cryoprotectants, surfactants, and antibioticagents (e.g., sodium azide, ciprofloxacine, chloramphenicol, gentamicin,amikacin, tobramycin, or amphotericin B).

The solution for analytes can be based on the composition andcharacteristics of the samples to be tested, e.g., a biological samplesuch as saliva, serum, plasma, lymph, urine, milk, mucus, CSF, celllysate, tissue culture supernatant, etc. For example, a solution for amultianalyte control for urinalysis would be based on the composition ofurine to minimize confounding differences, such as pH or saltconcentrations. In some embodiments, the solution is assembled frompurified components, either synthetic or biologically sourced, to mimicthe biological sample (e.g., 5% HAS in 0.9% PBS or 50 mM Tris bufferwith 150 mg/dL purified bovine cholesterol). In some embodiments, thesolution is derived from the biological sample and processed to removecomponents that will interfere with detection of the analytes, e.g.,analytes to be detected (e.g., in high endogenous amounts), fibrin,extraneous proteins, lipids, contaminants, or other components.Processing can include charcoal stripping to remove endogenous steroidhormones, heat treatment to reduce protease activity, or Celitetreatment to reduce lipids.

E. TYPES OF ASSAYS AND ANALYTES

Provided herein are compositions, kits, and methods for designingmultianalyte controls with increased stability. The multianalyte controlcan be used with any standard bioassay or assay format. The multianalytecontrol can be used to compare to and quantitate components ofbiological samples (e.g., saliva, serum, plasma, lymph, urine, milk,mucus, CSF, cell lysate, tissue culture supernatant) or other sampletypes, and monitor assay performance.

The presently described multianalyte controls can be used with anymultianalyte assays. Such assays have been designed for diagnosing orcharacterizing a number of conditions, e.g., urinalysis for kidney orliver function, anti-nuclear antibodies (ANA test), cancer (e.g.,bladder, prostate, ovarian cancer), dyslipidemia, Alzheimer's disease,atherosclerosis, diabetes, cardiac function, immune function, etc.

In some embodiments, the multianalyte control is for a multianalyteurine test. In some embodiments, stable control analytes for amultianalyte urine test include any one or more (1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, or all 13) of amylase, calcium, chloride, cortisol,glucose, hCG, magnesium, microalbumin, phosphorus, potassium, sodium,urea nitrogen, and uric acid in any combination. In some embodiments,stable control analytes for a multianalyte urine test further includeany one or more (1, 2, 3, 4, or all 5) of ketones, leukocyte esterase,nitrite, protein, and urobilinogen. In some embodiments, stable controlanalytes for a multianalyte urine test include any one or more (1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or all 37) of3-Methoxytyramine, 5-Aminolevulinic Acid, 5-HIAA,17-Hydroxycorticosteroids, 17-Ketogenic Steroid, 17-Ketosteroid,Aldosterone, AMP (Cyclic), Arsenic, Calcium, Chloride, Coproporphyrin,Cortisol, Dopamine, Epinephrine, Glucose, HVA, Hydroxyproline, Iron,Lead, Magnesium, Mercury, Metanephrine, Microalbumin, Norepinephrine,Normetanephrine, Phosphorus, Porphyrins, Potassium, Protein, Sodium,Urea, Urea Nitrogen, Uric Acid, Uroporphyrins, VMA, and Zinc, in anycombination.

In some embodiments, unstable analytes for a multianalyte urine testinclude creatinine. Creatinine is included in the Uric Acid, BUN,Amylase, Calcium, Magnesium, and Creatinine tests for detection on theBeckman Coulter DxC, and considered unstable relative to the otheranalytes in the tests. In some embodiments, unstable analytes for amultianalyte urine test include bilirubin. In some embodiments, unstableanalytes for a multianalyte urine test include creatinine and bilirubin.In some embodiments, unstable analytes for a multianalyte urine testinclude any one or more (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, or all 17) of 11-β-Hydroxy-Androsterone,11-β-Hydroxy-Etiocholanolone, 11-Ketoandrosterone,11-Ketoetiocholanolone, Androsterone, Barbiturate, Bilirubin, Copper,Coproporphyrin, Creatine, Creatinine, Cystine, Dehydroepiandrosterone,Estriol, Etiocholanolone, Hydroxyproline, Pregnanetriol, in anycombination.

Multianalyte controls are often packaged or used as multilevel controls,that is, where there are a plurality of tubes containing the sameanalyte(s) at different levels. A multianalyte control as describedherein can include a container holding at least one unstable controlanalyte in stablized form (unstable control level 1) and a containerholding at least one stable analyte in solution (stable control level1). A multilevel control would include additional containers, e.g.,unstable control level 2, and in some embodiments, unstable controllevel 3, and in some embodiments, unstable control level 4, etc. Themultilevel control can also include additional containers with, e.g.,stable control level 2, stable control level 3, and in some embodiments,stable control level 4.

In some embodiments, the levels of the multilevel controls are preparedto cover a range, e.g., a range of normal levels for the includedanalytes. For example, a normal range of sodium concentrations in a24-hour urine collection test is 40-220 mmol/day for an adult. Assumingan average urine output of 1 liter per day, an exemplary set ofmultianalyte controls can be prepared so that sodium will berepresentative in the assay of 40 and 200 mmol, optionally withintermediate levels (e.g., 120 mmol). A normal range of serum creatinineis 53-113 micromolar for an adult. An exemplary set of multianalytecontrols can be prepared so that creatinine will be representative inthe assay of 53 and 113 micromolar, optionally with intermediate levels.In some embodiments, the multilevel control includes containersrepresenting 2 or 3 levels that cover normal ranges of the includedanalytes, e.g., a low level multianalyte control representing the lowconcentration of the included analyte ranges, and a high levelmultianalyte control representing the high concentration of the includedanalyte ranges. In some embodiments, the multilevel controls areprepared to include normal and abnormal levels of the included analytes.For example, higher than normal urine cortisol can be indicative ofCushing's Syndrome; normal cortisol levels for a 24 hour urine sampleare usually <50 micrograms/day. Multilevel controls in this case can beprepared to represent, e.g., 20 micrograms, 50 micrograms, 100micrograms, and 200 micrograms. One of skill can determine normal orabnormal levels of a given analyte using information available in thefield, e.g., MedlinePlus from the NIH, the FDA, or the Mayo Clinicwebsite.

F. EXAMPLES 1. Example 1 Preparation of Lyophilized Analyte Spheres

To manufacture a composition of lyophilized unstable analyte,small-diameter beads (3-9 mm), each containing a precise and accurateamount of a concentrated liquid (25-250 uL) containing one or multipleunstable analytes were dispensed and lyophilized as spheres. To helpprepare consistent, stable, and durable spheres, excipients were used inthe formulation of the spheres. The spheres can be produced commerciallyand in large scales by BioLyph, LLC (Hopkins, Minn.).

Two solutions containing 8390 mg/dL or 8665 mg/dL of creatinine indeionized water were used to prepare two different creatinine beads withdifferent concentrations (low and high). These solutions were combinedwith additives, and then dispensed and lyophilized to produce the beads.The concentration of creatinine in the solution intended for productionof the creatinine spheres was significantly higher than theconcentration of creatinine control levels needed in order to accountfor the dilution factors due to addition of spheres to thereconstitution control fluid.

Table 1 and FIG. 1 show the characteristics and bead-to-bead variabilityof creatinine beads.

TABLE 1 Creatinine bead characteristics Bead Coeffi- Size Mass ofStandard cient of Diam- Dispense Mass of Analyte Deviation Varia- Beadeter Volume Bead in Bead (+/−) tion (%) Creatinine 5 mm  50 μL 7.27 mg8.39 mg 0.108 mg 2.77 (low level) Creatinine 6 mm 100 μL 15.3 mg 8.65 mg0.319 mg 3.69 (high level)

2. Example 2 Preparation of Quality Control with Stable Analytes

Formulation of the Urine Chemistry Control begins by processing thenormal human urine base. An enzymatic heat treatment is conducted todegrade endogenous labile creatinine. The urine is incubated with 100U/dL Creatininase and 50 U/dL Creatinase at 25° C. for 1 hour. The nextstep is a 7-day, 41° C. incubation. Following treatment, the processedurine is diafiltered to remove the degradative enzymes. Finally, thecollected urine is diluted with an equal volume of deionized waterbefore testing creatinine background concentration.

TABLE 2 Endogenous creatinine concentration in urine base before andafter processing Creatinine concentration Unprocessed urine 45.03 mg/dLCreatinine degraded urine 12.55 mg/dL

Following the initial processing, stable analytes and other matrixconstituents are added to the desired concentrations. The resultingcontrol will have the analytes in a traditional Urine Chemistry Controlexcept for creatinine, which can be added via lyophilized bead at thepoint of use. Urine chemistry controls can include any one or more ofamylase, calcium, chloride, cortisol, glucose, hCG, magnesium,microalbumin, phosphorus, potassium, protein, sodium, urea, ureanitrogen, and uric acid.

3. Example 3 Preparation of the Quality Control at the Point of Use

The quality control was prepared by rehydrating the appropriate numberof analyte beads (each with a defined and assayed concentration) in theappropriate volume of the quality control with stable analytes toachieve the desired concentration for each level of control. Multiplelevels of control with different concentration of analyte were preparedto achieve analyte concentrations at below, near, or above the clinicaldecision points of the assays. Analyte concentrations were thendetermined to ensure multi-level and clinical utility of thecomposition. The resulting compositions were then capped and stored at2-8° C.

In this example, a tri-level multi-analyte Urine Chemistry Control wasformulated. Tri-level creatinine utility was achieved by adding onelow-level creatinine bead to 5 mL of Level 1 control, one high-levelcreatinine bead to 5 mL of Level 2 control, and one of each levelcreatinine bead (2 beads total) to 5 mL of Level 3 control.

Table 3 shows the recovery data for analytes in a tri-levelmulti-analyte Urine Chemistry Control prepared at the point of use byreconstituting the lyophilized creatinine analyte sphere in a liquidurine chemistry composition containing stable analytes.

TABLE 3 Concentrations of analytes in the urine chemistry controlAnalyte Unit Level 1 Level 2 Level 3 Amylase U/L 80 211 289 Calciummg/dL 7.9 11.6 20.8 Chloride mmol/L 86 209 255 Glucose mg/dL 32 336 393hCG Qualitative − + + Magnesium mg/dL 4.6 11.2 20.5 Microalbumin mg/L 34127 184 Phosphorus mg/dL 26.7 49.3 76.7 Protein mg/dL 25 72 121 Sodiummmol/L 83.8 178.0 216.7 Urea Nitrogen mg/dL 474.1 745.9 844.5 Uric Acidmg/dL 17.0 22.0 27.0 Creatinine mg/dL 106.01 198.39 290.16

Stabilities of the analytes were evaluated by using an acceleratedstability model to predict shelf life. Vials of the liquid urinechemistry control containing stable analytes and vials of creatininebeads were stored at elevated temperatures (35, 40, and 45° C.) forpre-determined periods of time to observe analytedecomposition/degradation more rapidly than the recommended storagetemperature (2-8° C.). The samples were then assayed for analyteconcentration at the end of various incubation periods. The results showthat the analytes would be stable for at least 4 years when stored at2-8° C. Table 4 shows the real time stability data for creatinine forthe first 18 months of the study. Statistically significant results areindicated by italics.

TABLE 4 Real time stability of creatinine at 2-8° C. Traditional UrineChemistry Urine Chemistry Control Control of this Invention Time(month)Level 1 Level 2 Level 1 Level 2 Level3 1 0.27% −1.19% 0.73% 0.47% 1.71%6 −0.93% −3.70% 0.04% −1.17% 0.69% 12 −4.10% −8.08% −1.37% 0.87% 0.37%18 −8.79% −11.69% −1.80% −0.81% −0.55%

Open vial stability of the controls was also evaluated by simulatingactual laboratory use conditions. This was done by storing the vials ofcontrols prepared by adding creatinine beads at 2-8° C. and removingthem from the refrigerator every working day for 36 days, allowing thevials to equilibrate at room temperature for 15 minutes, opening thevials and exposing their contents to the laboratory environment, andclosing the vials and returning them to the recommended storagetemperature of 2-8° C. Samples of the vials were assayed during thisopen vial stability study for analyte concentration. The results of thisstudy indicate that all quantitative analytes from Table 3 will bestable for at least 36 days when prepared as described above and storedat 2-8° C.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All patents, patent applications, internetsources, and other published reference materials cited in thisspecification are incorporated herein by reference in their entireties.Any discrepancy between any reference material cited herein or any priorart in general and an explicit teaching of this specification isintended to be resolved in favor of the teaching in this specification.This includes any discrepancy between an art-understood definition of aword or phrase and a definition explicitly provided in thisspecification of the same word or phrase.

1. A multianalyte control kit comprising a first container holding atleast one unstable control analyte, wherein the at least one unstablecontrol analyte is lyophilized; and a second container holding at leastone stable control analyte.
 2. The multianalyte control kit of claim 1,wherein the at least one unstable control analyte is lyophilized in theform of beads.
 3. The multianalyte control kit of claim 1, wherein theat least one stable control analyte is in solution.
 4. (canceled)
 5. Themultianalyte control kit of claim 1, further comprising a containerholding a base matrix solution.
 6. (canceled)
 7. The multianalytecontrol kit of claim 5, wherein the base matrix comprises at least onecomponent selected from the group consisting of: protease inhibitor,chelating agent, buffer, salt, antioxidant (or antioxidant enzyme),cryoprotectant, surfactant, and antibiotic agent.
 8. The multianalytecontrol kit of claim 1, further comprising at least one additionalcontainer holding the at least one unstable control analyte at adifferent concentration than in the first container, and at least oneadditional container holding the at least one stable control analyte ata different concentration than in the second container. 9-10. (canceled)11. A method for assembling a multianalyte control kit comprisingselecting at least one unstable analyte in the multianalyte control kitand lyophilizing the at least one unstable analyte in a first container;and adding at least one stable analyte in the multianalyte kit to asecond container.
 12. The method of claim 11, wherein the at least oneunstable control analyte is lyophilized in the form of beads.
 13. Themethod of claim 11, wherein the at least one stable analyte is insolution. 14-15. (canceled)
 16. A method for preparing a multianalytecontrol kit for use in an assay comprising, resuspending at least oneunstable analyte in lyophilized form, and combining the at least oneunstable analyte with at least one stable analyte in solution.
 17. Themethod of claim 16, wherein the resuspending and combining steps aresimultaneous.
 18. The method of claim 16, wherein the resuspending andcombining steps are separate.
 19. The method of claim 16, wherein the atleast one unstable analyte and at least one stable analyte are added toa base matrix solution. 20-22. (canceled)
 23. A multianalyte control kitcomprising a container (1) holding creatinine lyophilized in the form ofbeads; and a container (2) holding at least one stable control analytein solution.
 24. The multianalyte control kit of claim 23, furthercomprising a container (3) holding creatinine lyophilized in the form ofbeads, wherein the amount of creatinine in containers (1) and (3) isdifferent.
 25. The multianalyte control kit of claim 23, furthercomprising a container (4) holding the at least one stable controlanalyte in solution, wherein the concentration of the at least onestable control analyte is different in containers (2) and (4).
 26. Themultianalyte control kit of claim 25, further comprising a container (5)holding creatinine lyophilized in the form of beads, wherein the amountof creatinine in containers (1), and if present (3), is different thanthe amount of creatinine in container (5).
 27. The multianalyte controlkit of claim 24, further comprising a container (6) holding the at leastone stable control analyte in solution, wherein the concentration of theat least one stable control analyte in containers (2), and if present(4), is different than the amount of the at least one stable controlanalyte in container (6). 28-29. (canceled)
 30. The multianalyte controlkit of claims 23, wherein the at least one stable analyte is in solutionof processed urine.
 31. The multianalyte control kit of claim 23,further comprising a container of processed urine.
 32. (canceled)